Nucleic acid hybridization assays are useful in the detection of particular nucleic acid sequences of interest, also referred to as "target" sequences. These target sequences may be characteristic of a particular disease-associated gene, or they may be specific for various organisms or cell types. Accordingly, detection and identification of a particular target sequence can provide diagnostically significant information.
The ability to detect target nucleic acid sequences in a sample by carrying out a hybridization reaction between a target nucleic acid and a complementary "probe" nucleic acid is the cornerstone of nucleic acid-based diagnostic technologies. These assays can generally be characterized as either "heterogeneous" or "homogeneous". Heterogeneous assays depend on the ability to separate hybridized from non-hybridized nucleic acids. One such assay involves immobilization of either the target or probe nucleic acid on a solid support so that non-hybridized nucleic acids which remain in the liquid phase can be easily separated after completion of the hybridization reaction (Southern, J. Mol. Biol., 98: 503-517 (1975).)
In comparison, homogeneous assays depend on other means for distinguishing between hybridized and non-hybridized nucleic acids. Because homogeneous assays do not require a separation step, they are generally considered to be more desirable. One such homogeneous assay relies on the use of a label attached to a probe nucleic acid that is only capable of generating signal when the target is hybridized to the probe (Nelson, et al., Nonisotopic DNA Probe Techniques, Academic Press, New York, N.Y., pages 274-310 (1992).)
One of the most significant obstacles to the development of nucleic acid-based diagnostic assays has historically been a lack of sensitivity. In particular, when the number of copies of the target nucleic acid in a sample are limited, sensitivity becomes very important. Many strategies have been designed to overcome this obstacle, with variable success. Among the most successful strategies are those that involve either target amplification or signal amplification. Target amplification involves increasing sensitivity by exponentially multiplying the number of copies of target sequences in a sample. Examples of target amplification techniques include the polymerase chain reaction, or "PCR" (Saiki, et al, Science 239: 487-491 (1988), and ligase chain reaction, or "LCR" (Wu, et al., Genomics 4: 560-569 (1990).)
Another method for increasing sensitivity is by amplifying the detectable signal which is generated by a single target/probe hybridization event. This can be accomplished by utilizing branched probes, each being capable of generating multiple detectable signals (Urdea, et al, Clin. Chem. 39: 725-726 (1993)), or by utilizing cycling probe technology, or "CPT", which relies on the ability to generate multiple detectable probes from a single target sequence (Bekkaoui, et al., BioTechniques, 20: 240-248 (1996).)
Still another method for increasing the sensitivity of nucleic acid-based diagnostics employs a cascade reaction to amplify signal. Libeskind (U.S. Pat. No. 4,699,876) discloses a heterogeneous assay utilizing a probe with an enzyme activator attached thereto. Once the probe binds to the target and unhybridized probe is removed, the enzyme activator is used to initiate a signal generation cascade which produces amplified signal levels.
The present invention provides for a homogeneous assay which employs a target cycling reaction, or "TCR", to provide for target amplification. This reaction involves the use of a target analog to mimic the presence of target nucleic acid in a sample. When coupled with a simultaneous signal amplification reaction, the present invention exhibits substantially enhanced sensitivity.